R. McCarty
M. Futai
K. Altendorf
W. Junge
Y. Mukohata
J. exp. Biol. 172, 431-441 (1992)
Printed in Great Britain © Tlie Company of Biologists Limited 1992
43 ^
A PLANT BIOCHEMIST'S VIEW OF H+-ATPases AND ATP
SYNTHASES
BY RICHARD E. McCARTY
Department of Biology, The Johns Hopkins University, Baltimore, MD 21218, USA
Introduction
My twenty-five year fascination with membrane ATPases grew out of my experiences
in the laboratories of Andre" Jagendorf and Efraim Racker. Andre" introduced me to
photosynthetic phosphorylation and Ef, to whose memory this article is dedicated,
convinced me that ATPases had much to do with ATP synthesis.
Astounding progress has been made in the H+-ATPase field in just two decades. By the
early 1970s, it was generally recognized that oxidative and photosynthetic ATP synthesis
were catalyzed by membrane enzymes that could act as H+-ATPases and that the
common intermediate between electron transport and phosphorylation is the
electrochemical proton gradient. At that time, it had been shown that a cation-stimulated
ATPase activity was associated with plasma membrane preparations from plant roots.
The endomembrane or vacuolar ATPases were unknown.
The application of improved biochemical methods for membrane isolation and
purification, as well as membrane protein reconstitutions, led rapidly to the conclusion
that there are three major classes of membrane H + -ATPases, P, V and F. P-ATPases,
which will not be considered further in this article, are phosphorylated during their
catalytic cycle and have a much simpler polypeptide composition than V- or F-ATPases.
The plasma membrane H+-ATPase of plant, yeasts and fungal cells is one example of this
class of enzymes (see Pedersen and Carafoli, 1987, for a comparison of plasma
membrane ATPases).
Biochemical and gene sequencing analysis have revealed that V- and F-ATPases
resemble each other structurally, but are distinct in function and origin. The ' V stands for
vacuolar and the 'F' for FiF 0 . Fi was the first factor isolated from bovine heart
mitochondria shown to be required for oxidative phosphorylation. F o was so named
because it is a factor that conferred oligomycin sensitivity to soluble Fi. Other F-ATPases
are often named to indicate their sources. For example, chloroplast Fi is denoted CF| (see
Racker, 1965, for early work on Fi). Recent successes in reconstitution of vacuolar
ATPase have led to a V| V o nomenclature for this enzyme as well.
The term 'ATP synthase' is now in general use to describe F-ATPases. This term
emphasizes the facts that although F-ATPases function to synthesize ATP, they do not
catalyze, normally, ATP hydrolysis linked to proton flux. In contrast, V-ATPases are very
unlikely to operate as ATP synthases. Thus, F-ATPases are proton gradient consumers,
whereas V-ATPases generate proton gradients at the expense of hydrolysis.
Key words: ATP synthesis, ATP hydrolysis, FiF0) ATP synthase.
432
R. E. MCCARTY
In this brief review, 1 will compare the structures of F- and V-ATPases. Also, I give
some insight into the mechanisms that help prevent wasteful ATP hydrolysis by the
chloroplast ATP synthase (CF|F 0 ).
Structure of F- and V-ATPases
Both F- and V-ATPases are structurally complex. CFiF 0 , for example, contains nine
different proteins and a total of about twenty polypeptide chains (Jagendorf et al. 1991).
The Mr of V- and F-ATPases is approximately 550000-650000. The complexity of these
enzymes is puzzling. This is especially true in view of the fact that P-ATPases can be as
simple as a single polypeptide chain of about 100 000Mr. Genetic studies with
Escherichia coli have established that each of the eight E. coli FiF 0 polypeptides is
required for function (Futai et al. 1989).
F-ATPases may be readily separated into two parts - Fi, the catalytic sector and F o , the
proton translocating part. Fi can be removed from many coupling membranes either by
physical means or simply by diluting membranes into media of low ionic strength that
contain EDTA. Fi is, thus, an extrinsic membrane protein. Once removed from the
membrane, Fi is soluble to at least lOOmgml" 1 . From 2 kg of spinach leaves, 400 mg of
CFi, greater than 95 % pure, may be prepared in a day and a half. This ease of preparation
makes one less reluctant to engage in calorimetric or equilibrium nucleotide binding
studies that can require as much as 100 mg of CFi for each experiment. Depending on the
source, the Mr of Fi ranges from 365 000 to 400000.
From all sources, mitochondrial, chloroplast and eubacterial plasma membranes, the
subunit composition of Fi is similar. There are five different proteins, labelled a-e in
order of decreasing MT. There is a consensus that these polypeptides are present in a
3:3:1:1:1 stoichiometry (Table 1). The primary sequence of a large number of Fi
polypeptides has either been deduced from gene sequences or determined chemically.
From sequence comparisons, three striking facts were revealed. First, the sequence of the
/3 subunit has been remarkably conserved, even across Kingdom lines (Hudson and
Mason, 1988; Walker and Cozens, 1986). For example, the /3 subunit of CFi and E. coli
F| have 76% sequence identity. Second, the a and /3 subunits are related and it seems
quite possible that a arose by gene duplication. Third, the smaller subunits, y, 8 and e, are
not well conserved. In fact, e of mitochondrial Fi does not resemble CFi e or E. coli Fi e
Table 1. Subunit composition of some ATP synthases
Source
a
E. coli
55
Chloroplasts
Mitochondria
55
55
Extrinsic polypeptides
Intrinsic polypeptides
Fi
Fo
S
e
a
b
c
50
y
31
19
15
54
52
36
30
21
20
15
15
30
IV
27
30
17
I
17
20
8
II
III
14
8
8+at least five
polypeptides
Numbers shown are Mr valuesXlO" 3 . (Adapted from Nelson, 1989.)
H+-ATPases and ATP synthases
433
at all and mitochondrial Fi 5 is also unique (Walker and Cozens, 1986). The bacterial and
chloroplast Ssubunits resemble oligomycin sensitivity conferral protein (OSCP) which is
considered to be an F o subunit. Thus, generalizations about subunit function based simply
on Mr can be problematic.
From image reconstruction electron microscopy (Boekema et al. 1988), distance
mapping by fluorescence energy transfer (McCarty and Hammes, 1987) and X-ray
diffraction (Bianchet era/. 1991), the structure of Fi is emerging (Fig. 1). The structure is
dominated by the a//3 hexamer. The a subunits are in contact with the /3 subunits, but the
a subunits are closer to the membrane surface. The position of the y subunit is not exactly
known, but it is likely that it is centrally located in an asymmetrical manner. The e subunit
interacts strongly with y, and 8 is placed close to the membrane surface because of its role
in Fi binding to F o .
The inherent structural asymmetry of Fi is intriguing. The a and /3 subunits cannot be
in equivalent environments. That is, the interactions of a given a or (3 subunit in the
complex with the smaller subunits must differ from those of another a and /3 subunit.
There is evidence to suggest that CFi lacking the S and e subunits retains its structural
asymmetry (Shapiro et al. 1991). Thus, interactions of the y subunit with a and /3 may be
the major asymmetry inducers.
ADP+P,
ATP
Lumen
Fig. 1. A depiction of the chloroplast ATP synthase (CF|FO). The model of CFi is supported
by electron microscopy and image reconstruction, fluorescence resonance energy transfer
distance mapping of specific residues on various CFi subunits and the arrangement of the a
and /3 subunits is in accord with the X-ray diffraction results for mitochondrial Fi. The
arrangement of CF 0 subunits is entirely speculative. Courtesy of Dr Carolyn Wetzel.
434
R. E. MCCARTY
The structural asymmetry of Fi probably explains the nucleotide binding site
asymmetry as well as the unusual reactivity of Lys-378 of the a subunit of CFi. Only one
of the three a subunits of CFi reacts at this position rapidly with Lucifer Yellow vinyl
suLfone. The six nucleotide binding sites of Fi show quite different properties. The most
plausible model for the mechanism of Fi is one in which two or three catalytic sites
change their properties during the catalytic cycle (Boyer, 1989). In this alternating site or
binding change mechanism, catalytic sites alternate between very high affinity and much
lower affinity. The binding of substrate to one catalytic site promotes catalysis and
product release from another. If this mechanism holds, and there is much evidence in its
favor, Fi cannot be permanently frozen in one asymmetrical state. The incubation of CFi
with Mg 2+ -ATP was found to cause two nucleotide binding sites on CFi to switch their
properties (Shapiro and McCarty, 1990). This observation is consistent with the binding
change mechanism. How substrate binding affects changes in the enzyme is unknown.
Although all five E. coli ¥\ or CFi subunits are required for ATP synthesis, 03/337
complexes exhibit the highest ATPase activity. An intact y subunit, however, is not
required for ATPase activity and some ATPase activity is seen in o//3 complexes (see, for
example, Kagawa et al. 1989). Although it probably bears the catalytic sites, isolated /3 is
a very poor ATPase. In CFi, y definitely plays a regulatory role and has been proposed to
be involved in proton translocation. The 8 subunit is required for the binding of E. coli Fi
to F o and is necessary for the functional binding of CFi to CFO. In E. coli and chloroplast
Fi, e is an inhibitory subunit, probably involved in regulation of the enzymes. These
studies are summarized by Futai et al. (1989) and Jagendorf et al. (1991).
Different coupling membranes seem to have tailored the F o portion of the ATP
synthase to suit their needs. By definition, F o is that part of the ATP synthase that remains
after removal of Fi. Unlike Fi, F o is hydrophobic and can only be isolated from
membranes by detergent extraction. E. coli F o (Deckers-Hebestreit and Altendorf, 1992)
and chloroplast F o have been purified in active forms. F o functions in at least two ways: it
binds Fi and translocates protons across the membrane. The rates of proton conductance
through CF 0 are high, suggesting that the F o proton translocation mechanism is a protonselective channel (Lill and Junge, 1989; Junge et al. 1992).
The number of different polypeptides in F o varies with the source (Table 1). E. coli F o
is the simplest, with just three polypeptides, labeled a, b and c. Spinach CF 0 has four
polypeptides, denoted in Roman numerals, I-FV. Mitochondrial F o can have as many as
eight polypeptides. All F o subunits contain a small (8000MT) hydrophobic polypeptide,
referred to as the proteolipid or DCCD-binding protein. There is no lipid covalently
attached to the DCCD-binding protein. This polypeptide is present in 6-12 copies per
ATP synthase. Remarkably, the reaction of just one copy of the protein with DCCD
(A^W-dicyclohexylcarbodiimide) at a specific glutamyl or aspartyl residue is sufficient to
inhibit ATP synthesis totally. ATP synthesis is inhibited by DCCD because proton
transport through F o is blocked. All three E. coli F o subunits are required for proton
translocation and at least subunits III and IV of chloroplast F o are necessary for proton
transport.
Cross-linking studies indicate that several CFO subunits interact with CF|. Some CF 0
subunits have hydrophilic domains that are predicted to extend out from the stromal side
H+-ATPases and ATP synthases
435
of the membrane; that is, towards CFi. Subunit HI of CF 0 appears to bind to CFi with
sufficient strength to allow the purification of a CFi-subunit HI complex by a
chromatographic procedure (C. Wetzel and R. McCarty, unpublished observations). This
complex retains some of the properties characteristic of the CFiF 0 from which it was
prepared. Probably, all CF 0 subunits are in contact with one or more CFi polypeptides.
The dominant interactions have yet to be determined.
Like F-ATPases, V-ATPases consist of an extrinsic component, called Vi, and a
membrane-associated part, now denoted Vo. The treatment of yeast, Neurospora crassa,
or oat root vacuolar membranes and clathrin-coated vesicles with KI or KNO3 in the
presence of Mg 2+ -ATP rapidly inactivates ATPase activity and associated proton
pumping. Depending on the source, about five polypeptides are dissociated from the
membrane by this treatment (see, for example, Ward et al. 1992). The dissociated
polypeptides were devoid of ATPase activity by themselves, but, in the case of the
clathrin-coated vesicles (Puopolo et al. 1992) and oat root systems (Ward et al. 1992),
could be added back to the depleted membranes to give both ATPase activity and ATPdependent proton pumping. It is of interest to note that membranes from which the Vi
components had been removed were not leaky to protons. Thus, either V o required a Vi
polypeptide(s) for proton transport or the attachment of Vi helps to open a proton gating
mechanism.
To an F1 person, the subunit composition of V-ATPases is somewhat confusing. Then
again, to a Vi person, I would venture to guess that the composition of F-ATPases would
also be confusing. As is the case for F-ATPases, V-ATPases seem to retain a similar
subunit structure for Vi, but tailor the membrane-associated components to fit the needs
of the membrane (Nelson, 1989). The situation is made even more complicated by the
possibility that a given tissue within an organism could possess a V-ATPase that differs
from that in another tissue.
Regardless of the apparent heterogeneity of the subunit composition of V-ATPases,
there are, as is the case for F-ATPases, recurrent themes. V-ATPases are oligomeric and
contain both a peripheral, catalytic component Vi and an integral membrane part, called
V o by analogy to F o (Table 2). All V-ATPases so far examined from animal, plant, fungal
and yeast sources contain two easily discernable polypeptides of about 70000 (the A
subunit) and 60000M r (the B subunit). Based on covalent binding of -SH alkylating
reagents and photoaffinity nucleotide binding, it is very likely that the A subunit is
catalytic. The function of the B subunit is unknown. The A subunits of various VATPases share remarkable amino acid sequence similarity. Remarkably, the catalytic
subunits of ATP synthases of archaebacterial ATPases are more closely related to VATPases than to F-ATPases. Nonetheless, the A subunits of both archaebacterial and VATPases share significant sequence homology with the /3 subunits of Fi (22-26 %) (Ihara
et al. 1992). Moreover, the B subunits of V-ATPases are homologous with the a subunits
of Fi (20-27%). See Nelson (1992) for a more complete description of the sequence
homologies.
In addition to subunits A and B, V-ATPases from a number of membranes contain
polypeptides ranging in relative molecular mass from 100 000-115 000 to 12 000-13 000.
Most V-ATPases contain polypeptides (four or five) in the 30000-45 000 Mr range. All
436
R. E. MCCARTY
Table 2. Subunit composition of some V-ATPases
Extrinsic polypeptides*
Vi
Intrinsic polypeptidest
Source
Chromatin granules
Yeast
Oat root
Red beet
Archaebacterial
73
69
70
67
64
B
C
D
E
58
60
60
55
54
40
42
44
52
28
34
36
41
44
33
32
36
42
29
32
100
100
16
29
19
27
13
16
9
17
17
12
'Defined by their ease of removal from vacuolar membranes by chaotropic anion treatment in the
presence of Mg2+-ATP. The numbers are Mr values (xl0~ 3 ) of the peptides.
^Subunits left behind after chaotroph/ATP treatment. The numbers are Mr values (x!0~ 3 ) of the
polypeptides.
so far contain a 16000 MT protein that has the solubility properties of a 'proteolipid' and
binds DCCD. As is the case for F-ATPases, DCCD inhibits V-ATPase activity and
associated proton pumping. It is quite possible that the V-ATPase DCCD-binding protein
arose by gene duplication. Some V-ATPases appear to contain a high Mr
(100 000-115 000) polypeptide and others a low Mr (12 000-13 000 MT) polypeptide.
V-ATPases are sensitive to inhibition by chaotrophic anions (I~, NO3" or SCN~) in a
manner that is fascinatingly dependent on the presence of Mg 2+ -ATP) (Moriyama and
Nelson, 1989). A number of polypeptides dissociate from the membrane as a result of this
treatment; others are left behind. Concomitant with loss of ATPase activity and
associated protein pumping, more or less spherical particles of 10-12 nm are removed
from vacuolar membranes (Bowman et al. 1992).
The peripheral portion (Vi) is defined as that part removed by chaotroph treatment in
the presence of Mg 2+ -ATP. The A and B subunits, probably present in a 3:3 stoichiometry
with respect to the smaller polypeptides, are clearly in this class. In addition, the
polypeptides in the 30000-45 000Mr range are removed by this treatment. Recently yeast
subunit C (Afr 42000) was cloned and sequenced (Beltran et al. 1992). Subunit C was
shown to be required for assembly and exhibited no homology to the y subunit of Fi.
What is left after chaotroph treatment is defined as V o . There is a consensus that the
16 000M r DCCD-binding protein, which is probably present in multiple copies, is part of
Vo. The approximately 100000Afr polypeptide of some V-ATPases as well as smaller
proteins (12 000-20000M T ) may also be part of Vo. Further analysis is required and will
undoubtedly be forthcoming.
There are several observations that point to major differences between V- and FATPases, even though superficially they seem similar. Removal of Fi greatly increases
the proton permeability of the membrane in which it resides. This effect is not observed
for V-ATPases. Perhaps some Vi component remains associated with V o after chaotroph
treatment and blocks a proton conductance mechanism. Alternatively, a Vi component is
required for proton transport through Vo. So far Vi has not been released from the
H+-ATPases and ATP synthases
437
membrane in a form that is active in ATP hydrolysis. This result is in distinct contrast to
FiF 0 . Finally, Mg 2+ -ATP destabilizes V|V 0 , whereas it stabilizes ¥\ against coldinactivation in the presence of chaotrophic anions.
Regulation of CFiF 0
The reaction catalyzed by the chloroplast ATP synthase is:
3Hfn+ADP+Pi •-
3Hout+ATP+H2O,
where 'in' and 'out' refer to the thylakoid lumen and stromal spaces, respectively. Logic
dictates that CF|F 0 should be able to catalyze the reverse reaction of ATP synthesis, ATP
hydrolysis. Within chloroplasts, however, it is very unlikely that CF|F 0 catalyzes ATP
hydrolysis at significant rates, even though it carries out ATP synthesis at very rapid rates.
Thylakoid membranes, as usually isolated, hydrolyze Mg 2+ -ATP in the dark, even at
37 "C, at rates close to 0.05 /nmol min~' mg~' protein. Much of this activity may be
attributed to phosphohydrolases that contaminate the preparations. In contrast, the same
membranes, when illuminated, can catalyze ATP synthesis at rates in excess of
5 ^unol min~' mg~' protein. It is clear, then, that light dramatically activates the
chloroplast ATP synthase. The ATP synthases have evolved to be specialists in ATP
synthesis and, through a number of different mechanisms, potentially wasteful ATP
hydrolysis by CF|F 0 is prevented.
It must be emphasized that the effect of light is indirect. Transthylakoid
electrochemical proton gradients drive ATP synthesis in total darkness and, thus, are also
involved in the conversion of the ATP synthase to an active form. Light-driven electron
flow generates proton gradients that serve two functions in photophosphorylation. First,
the proton gradient is the driving force for ATP synthesis and, second, it is required for
activation.
How this unusual 'one way' regulation occurs at the molecular level is beginning to be
revealed. A coherent picture, summarized in Fig. 2, of what may happen in a dark-to-light
transition can now be drawn. The e subunit is an inhibitor of the ATPase activity of CFi.
FAST
SLOW
ADP
CFyss-e
<Sy CF-yss^e ^ ^> CFTSH~E
ApH
Inactive
TSS TSH
Active
More active
Fig. 2. A scheme for the activation of the chloroplast ATP synthase. CF stands for the CFi part
of the ATP synthase. 7SS and 7SH are oxidized (disulfide) and reduced (dithiol) forms of the y
subunit, respectively. Tss and TSH are oxidized and reduced thioredoxin, respectively. In the
absence of ApH, CFi contains tightly bound ADP, and e and y interact strongly, as indicated
by the bold arrows. Rapid changes in die enzyme induced by ApH formation by electron
transport in the light cause ADP release and weaken y-e interactions (as shown by ~).
Although the oxidized enzyme in this state is active, further activation occurs upon reduction
of the y disulfide by reduced thioredoxin.
438
R. E. MCCARTY
By necessity, it must also inhibit ATP synthesis. The e and y subunits interact and it is
probable that the inhibitory effects of e are mediated at least in part through the y subunit.
In the dark, e interacts very strongly with y and the ATPase activity is inhibited.
Generation of the electrochemical proton gradient causes changes in CFi that involve
both the y and e subunits (at least). A movement of e relative to y is indicated. In their
new conformations, e inhibition could be nullified.
Redox regulation is also involved in the physiologically significant light activation of
CFi in chloroplasts. The y subunit of CFi from photosynthetic eukaryotes contains an
insert of about twenty amino acids that is absent in E. coli or mitochondrial Fi subunits
(Jagendorf et al. 1991). This insert contains a redox-active pair of Cys residues (Cys-199
and Cys-205 for spinach CFi y subunit). In the dark, Cys-199 and Cys-205 form a
disulfide bond, whereas in the light this disulfide (the only disulfide in CFi) is reduced.
Thioredoxin, reduced by electrons from photosystem I, is probably the physiological
reductant.
Although oxidized CFi is active in ATP synthesis, reduced CFi is even more active. At
physiological values of the electrochemical proton gradient, phosphorylation could be
enhanced as much as tenfold (Quick and Mills, 1986). Essentially, it appears that the
energy cost for activation is significantly decreased by reduction of the y disulfide bond.
The activated state of oxidized CFi in thylakoids decays quite quickly in the dark.
Thus, after a period of illumination, little ATPase activity is observed in the dark. After
reduction of the y disulfide bond, however, significant ATPase activity persists in the
dark after a period of illumination (Bakker-Grunwald, 1977). This ATPase activity is
coupled to inward proton fluxes and can generate ApH values of sufficient magnitude to
permit ATP synthesis at a relatively low rate. Proton ionophores at concentrations that
collapse the proton gradient completely abolish ATPase activity in the dark. The ApH
generated by ATP hydrolysis in the dark by CFi in reduced thylakoids is, thus, sufficient
to keep the enzyme, at least in part, in an active form. During illumination, ATP
hydrolysis is prevented by the high value of ApH which drives the reaction in the
direction of synthesis (Davenport and McCarty, 1986).
In intact chloroplasts, the ability of reduced CFi to hydrolyze ATP in the dark after a
period of illumination is lost over a period of several minutes in a biphasic manner. The
more rapid phase is probably a consequence of the binding of ADP to a nucleotide
binding site on CFi. ADP at micromolar concentrations rapidly inactivates the ATPase of
reduced CFi in the dark, without causing oxidation of the y subunit, dithiol. To reconvert
the CFi to an active form after ADP has bound, ApH is required and ADP is released from
the enzyme. The second, slower phase of the inactivation of CFiF 0 in intact chloroplasts
in the dark is the oxidation of the dithiol by an unknown mechanism (Biaudet etal. 1988).
There is good evidence that the e and y polypeptides of CFi interact. They are
physically close together (McCarty and Hammes, 1987) and alterations of the y subunit
modify e-CFi interactions (Soteropoulos et al. 1992). Reduction of the y disulfide
decreases the affinity of e binding to CFi about 20-fold and tryptic cleavage of a portion
of the C terminus of y abolishes high-affinity e binding. Removal of the e subunit
markedly enhances the rate of reduction of y disulfide by dithiothreitol or thioredoxin
(Dann and McCarty, 1992). The y subunit in CFi depleted in e is easily attacked by
H+ -A TPases and A TP synthases
439
proteases, with the initial major cut occurring close to the y disulfide to produce a y
fragment of about 27000A/ r . If the y disulfide is reduced, a second cut occurs that
releases a peptide of Mr 1300. This peptide bears Cys-205.
The y subunit of CFi in thylakoids in darkness is resistant to proteolysis. With the
increase of ApH in the light, however, changes in the enzyme occur that render y very
susceptible to partial proteolysis. The y subunit is cleaved to a fragment of 27 000 Mr and,
if the disulfide is reduced, the same second cut that occurs in CFi deficient in e is
observed. Thus, with respect to the susceptibility of the y subunit to protease attack, CFi
in illuminated thylakoids resembles CFi deficient in e.
Mitochondrial FiF 0 is regulated by a quite different mechanism from that of CFiF 0 .
The e subunit of mitochondrial Fi has no sequence similarity to that of CFi or E. coli F\.
Mitochondrial e does not appear to be an inhibitor of the ATPase activity of
mitochondrial F|. Instead, mitochondrial Fi activity is regulated by an inhibitor protein
(or proteins) that, unlike e, is not considered to be part of Fi (reviewed by Cross, 1981).
The binding of the inhibitor protein is favored by ATP. In contrast, the inhibitor protein is
released from the complex when the magnitude of the electrochemical proton gradient
and the ADP/ATP ratio are high. Thus, ATP synthesis would be favored.
There is no evidence for an inhibitor protein other than e in either E. coli or
chloroplasts. Moreover, it is very clear that e dissociation from CFi cannot be a part of the
activation process. CFi deficient in e binds to CF 0 equally as well as CFi, but e-depleted
CFi cannot restore ATP synthesis (Richter et al. 1984). When CFi is removed from
thylakoid membranes the membranes become highly proton permeable because of proton
leakage through CF 0 . The permeability is so high that even extremely rapid proton
translocation by light-dependent electron transport does not generate a significant ApH.
CFi deficient in e fails to block the CF 0 proton channel and, thus, cannot restore ATP
synthesis to CFi-depleted thylakoids. If e were to dissociate from CFi during activation,
therefore, ATP synthesis would be inhibited.
The e subunit is also an inhibitor of the ATPase activity of E. coli Fi and has been
shown to interact strongly with y (Dunn, 1982). The E. coli ATP synthase can operate as
an H + -ATPase in vivo. For example, when respiratory chain activity is low, ATP
hydrolysis by ATP synthase powers proton efflux to generate an electrochemical proton
gradient. It is likely that the activity of the enzyme is regulated since the ATPase activity
of the E. coli ATP synthase in its natural environment is much lower than the Vmax- An
energy-dependent activation mechanism which involves the overcoming of e inhibition
may occur in E. coli FiF 0 as well as in other ATP synthases.
The dual activities of the e subunit of CFi (regulation and proton channel blocking)
suggest an intriguing connection between activation and opening of a 'proton gate'. The
proton conductivity through CFiF 0 is normally low when the enzyme is inactive, but is
high (greater than 1 ms~') during ATP synthesis. Could it be that activation entails - at
least in part - an opening of the proton gate? In which part of the ATP synthase protons
induce the conformational changes involved in activation and proton gate opening is
unknown. Protonation of a CF 0 subunit(s) in contact with CFi could induce the changes.
Alternatively, CF 0 could act passively to deliver protons to a site on one or more of the
CFi subunits.
440
R. E. MCCARTY
To date, little is known about regulation of V-ATPases. By analogy to the FiF 0 ATPases, I predict that one or more of die smaller polypeptides of V i will be a regulatory
subunit. As is the case for mitochondrial Fi, a dissociable inhibitory protein could be
involved.
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